Primary Cell Culture: Frequently Asked Questions (FAQs)
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- Upon receiving the package, cryopreserved cells should be immediately transferred from the dry ice shipping container to a liquid nitrogen storage tank.
- In the unlikely event that no dry ice is left in the package upon receipt, thaw and use the cell immediately.
- Please note that storing the cells at -80°C can cause irreversible damage to them.
- Thaw the cryovial of cells in a 37°C water bath. It usually takes about 1-2 minutes.
- Take the cryovial out of the water bath when there are still some small ice crystals remaining in the vial.
- Prolonged exposure to heat will damage the cells, causing them not to plate.
- Do not spin down the cells to remove DMSO after thawing because the centrifugation process can cause even more cell damage than DMSO. Once the cells are plated in the recommended amount of media, the DMSO should be sufficiently diluted to prevent any immediate harm to the cells.
- Do not thaw the cells on the bench top at room temperature.
- After thawing and plating the cryopreserved cells, the first medium change should be done after 24 hours or overnight, so that both residual DMSO and any dead cells are removed.
- Thereafter, the medium should be changed every 48 hours until the cells are ready to be passaged.
- Cell Applications uses the term “population doubling” instead of “passage” for describing the growth potential of the cells.
- A population doubling is a two-fold increase in the total number of cells in culture. A passage is the propagation of a cell population by subculturing from one vessel to 3 or 4 vessels.
- Because different researchers use different split ratios when they subculture, it is difficult to predict how many passages can be obtained with a particular primary cell.
- Cell Applications primary cells are guaranteed for a specified number of population doublings (as indicated by product literature) when Cell Applications’ cell-specific growth media are used for the culturing procedures.
The following equations can be used to determine the maximum number of culture vessels that can be set up:
Total Cell #
Seeding Cell Density = cells
cells/cm2 = Surface Area (cm2)
Seeding Cell Density = cells
cells/cm2 = Surface Area (cm2)
Surface Area (cm2)
Growth Area of selected TC Ware (cm2) = # of tissue culture vessels that can be seeded
Growth Area of selected TC Ware (cm2) = # of tissue culture vessels that can be seeded
The effective growth areas for common tissue culture ware are listed below
| Culture Flasks | T-25 | T-75 | T-150 | T-250 |
|---|---|---|---|---|
| Growth Area | 25 cm2 | 75 cm2 | 150 cm2 | 250 cm2 |
| Culture Dishes | 35 mm | 60 mm | 100 mm | 150 mm |
|---|---|---|---|---|
| Growth Area | 9.6 cm2 | 20.4 cm2 | 57 cm2 | 143 cm2 |
| Multiwell Plates | 6 well | 12 well | 24 well | 96 well |
|---|---|---|---|---|
| Growth Area | 9.6 cm2 | 3.5 cm2 | 1.9 cm2 | 0.33 cm2 |
- No, our complete growth media are fully supplemented and ready-to-use for your convenience.
- Each complete medium contains all the essential components for optimized cell growth.
- Yes, we also offer growth media kits that consist of the basal media and growth supplements packaged separately.
- These kits are provided with instructions for preparation and storage.
- Yes, we may be able to provide custom media to accommodate your specific needs. For custom media orders additional charges may apply.
- Please contact us with your specific inquiries.
- The complete, fully supplemented, growth media is stable for 6 months at 4°C.
- The basal media is stable for 1 year at 4°C.
- The growth supplements are stable for 4 days at 4°C or are stable for 1 year at -20°C.
- Yes, Cell Applications’ Cytofect™ Transfection Kits deliver DNA to primary cells.
- Researchers have found that this cost-effective, uniquely formulated transfection reagent yields superior efficiency and lower toxicity when compared to transfection reagents from other manufacturers.
- For your convenience, Cytofect™ Transfection Kits include Cytofect™ tranfection reagents, enhancers, transfection media, and growth media.
- Most cells we offer can be plated on standard tissue culture grade ware.
- However, some cell types require coating material.
- Please see the respective cell product web pages for information on substrate reagents offered by Cell Applications
- All reagents should be at room temperature or below. Do Not Warm Reagents to 37°C.
- Do not over-trypsinize. Strictly follow the trypsinization instructions in the protocol.
- Watch cells under a microscope during the entire trypsinization process.
- When the cells become rounded but are still attached, hit the side of the flask against your palm until the cells detach. (If rounded cells detach by themselves without hitting, it means the cells are over-trypsinized.)